Thymidylate synthetase, the target enzyme for fluorouracil, forms a very stable complex requiring the drug metabolite, fluorodeoxyuridinemonophosphate, and the cofactor, methylenetetrahydrofolate. It is proposed that this complex can be used to entrap and thus stabilize endogenous methylenetetrahydrofolate from mammalian tissue. One of the important classes of cancer chemotherapeutic drugs, antifolates, are presumed to function through denial of the availability of methylenetetrahydrofolate and ultimately DNA synthesis. To date, however, there has been no means to directly and accurately measure levels of methylenetetrahydrofolate in cell extracts. We propose to develop the necessary methodology to fully characterize mammalian methylenetetrahydrofolate and other naturally occurring folates through incorporation into the thymidylate synthetase complex. To do so we must (1) establish conditions for extraction and complexation of endogeneous methylenetetrahydrofolate which prevent or at least minimize interference from other intercellular folates or folate binding proteins, (2) establish conditions to specifically convert other folate pools to the methylenetetrahydrofolate pool for caracterization, (3) identify electrophoretically the polyglutamates of methylenetetrahydrofolate and other interconvertible folates, (4) establish assay conditions and measure levels of both polyglutamate synthetase and hydrolase, (5) use these methodologies to characterize the folate status of both normal mouse liver and cultured mouse hepatoma cells which have been treated with cancer chemotherapeutic agents, such as methotrexate and fluorouracil.